A highly conserved A-to-I RNA editing event within the glutamate-gated chloride channel GluClα is necessary for olfactory-based behaviors in Drosophila

A-to-I RNA editing is a cellular mechanism that generates transcriptomic and proteomic diversity, which is essential for neuronal and immune functions. It involves the conversion of specific adenosines in RNA molecules to inosines, which are recognized as guanosines by cellular machinery. Despite the vast number of editing sites observed across the animal kingdom, pinpointing critical sites and understanding their in vivo functions remains challenging. Here, we study the function of an evolutionary conserved editing site in Drosophila, located in glutamate-gated chloride channel (GluClα). Our findings reveal that flies lacking editing at this site exhibit reduced olfactory responses to odors and impaired pheromone-dependent social interactions. Moreover, we demonstrate that editing of this site is crucial for the proper processing of olfactory information in projection neurons. Our results highlight the value of using evolutionary conservation as a criterion for identifying editing events with potential functional significance and paves the way for elucidating the intricate link between RNA modification, neuronal physiology, and behavior.

The PDF file includes: Figs. S1 to S5 Legends for tables S1 to S6 Table S7 Other Supplementary Material for this manuscript includes the following: Tables S1 to S6 Supplementary Figure 1.Location of the I27V editing site in the Drosophila GluClα subunit (UniProt ID Q94900).(A) SignalP 6.0 prediction showing that the probability of the presence of a signal-peptide cleavage site (CS) between positions 22 and 23 is P = 0.98.The Secretory (Sec) signal peptide n-region (Sec/SPI n) corresponds to the n-terminal region of the signal peptide and is labeled as N (above the amino acid sequence).Sec/SPI h corresponds to the center hydrophobic region of the signal peptide and is labeled as H. Sec/SPI c corresponds to the c-terminal region of the signal peptide and is labeled as C. OTHER corresponds to a sequence other than a signal peptide and is labeled as O. I27 is indicated by a green arrow.(B) PSIPRED 4.0 secondary structure prediction for the Drosophila GluClα immature protein.The full-length protein sequence was submitted for the prediction, whereas only the first 100 amino acids are shown.The protein secondary structure prediction is based on position-specific scoring matrices (PSSMs).Helices (H), strands (E) and coils (C) are indicated in pink, yellow and gray colors, respectively.Conf, confidence of prediction with the highest confidence in dark blue.AA, the target sequence.I27 is indicated by a green arrow.Note that transmembrane (TM) protein topology prediction based on support vector machines (SVM) (MEMSAT-SVM in the PSIPRED server) predicted amino acids 2-20 of the immature protein to be a signal peptide α-helix (not shown).(C) Iterative multiple sequence alignment between the Drosophila GluClα subunit (UniProt ID Q94900) and various subunits of anionic Cys-loop receptors whose three-dimensional (3-D) structure was determined at high resolution by X-ray crystallography or cryoelectron microscopy.For clarity, merely an N-terminal portion of the sequences is presented.The UniProt names and identifiers are indicated on the left side of the sequences.The PDB ID codes that correspond to the 3-D structures of the recombinant Cys-loop anionic receptors are indicated in square parentheses on the right side of the sequences.Signal peptides are underlined in red according to the UniProt Knowledgebase (UniProtKB).The edited amino acid (I27) is boxed in green and indicated by a green arrow.The first amino acid of each 3-D structure is boxed in cyan.Helix α1 is shown in white letters on pink background.The highly conserved aminoacid cluster mentioned in the manuscript text is indicated by a gray background.The beginning of the first beta strand (Ω1) is indicated by a yellow background.Asterisk indicates positions which have a single, fully conserved residue.Colon indicates conservation between groups of strongly similar properties.Period indicates conservation between groups of weakly similar properties.

Walk
Fly moves.

Stop
Fly is still.

Turn
Changes in fly's direction.

Touch
Fly actively touches another fly.

Social cluster
Fly sits in a group of 3 or more flies.

Long distance Approach
Fly approaches from a distance another fly and perform interaction.

Short distance approach
Fly performs an interaction during when in social cluster.

Chase
Fly chases another fly.

Chain
Chase with 3 or more flies.

Song
Fly moves one wing next to another fly.
Behavior bout length Length of the longest sequence of frames in which the behavior occurred per fly.
Behavior Modularity by length of interactions (LOI) Representation of how much the network is divided into modules according to interactions' length.
Strength by length of interactions (LOI) Length of interactions of a certain fly.
SD Strength according to length of interactions (LOI) Standard divination of the strengths according to interactions' length of flies from the same movie.
Betweenness Centrality by length of interactions (LOI) A measure of centrality of a certain fly based on shortest paths according to interactions' length.
SD Betweenness Centrality by length of interactions (LOI)